SNP molecular marker related to cadmium accumulation trait of wheat and breeding application thereof

Abstract

This invention discloses SNP molecular markers related to cadmium accumulation traits in wheat and their breeding applications, belonging to the field of gene biotechnology. The technical problem this invention aims to solve is how to rapidly and accurately identify cadmium content traits in wheat. To this end, it provides SNP loci TaCd2-1, TaCd2-2, and TaCd3, along with their detection substances. The average cadmium content of wheat samples with the genotype GGCCAA at these three SNP loci (TaCd2-1, TaCd2-2, and TaCd3) is significantly higher than that of other genotypes. The method provided by this invention offers a genetic basis for the rapid screening of high-cadmium-risk wheat germplasm and for early warning of high cadmium risk in wheat cultivation on cadmium-contaminated land, possessing significant theoretical value and application prospects for ensuring my country's food security.

Description

Technical Field

[0001] This invention belongs to the field of gene biotechnology, specifically relating to SNP molecular markers related to the cadmium accumulation trait in wheat and their breeding applications. Background Technology

[0002] Cadmium is a highly toxic trace element, classified as a Group 1 carcinogen. Wheat is one of my country's important staple crops, but due to the influence of its growing environment and ecosystem, cadmium-contaminated wheat poses a significant potential threat to human health and is a major hidden danger to my country's food security. Therefore, controlling the cadmium content in wheat grains is an urgent need to ensure food security and public health.

[0003] Wheat is a crop with relatively high cadmium accumulation, posing a certain risk of exceeding standards in cadmium-contaminated land. However, the physiological and molecular mechanisms of cadmium accumulation in wheat are still in their early stages. Furthermore, due to the complexity of the wheat genome and the diversity of wheat varieties, research on cadmium accumulation in wheat is challenging, and systematic studies on wheat varieties with low cadmium accumulation have not yet been conducted. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to quickly and accurately identify the cadmium content trait in wheat using molecular markers.

[0005] To address the aforementioned issues, this invention provides an application for detecting polymorphisms, genotypes, or haplotypes of SNPs in the wheat genome. The SNPs can be TaCd2-1, TaCd2-2, and TaCd3. TaCd2-1 is an SNP in the wheat genome, corresponding to position 320 of SEQ ID NO: 7, and is either C or G. TaCd2-2 is an SNP in the wheat genome, corresponding to position 233 of SEQ ID NO: 8, and is either C or G. TaCd3 is an SNP in the wheat genome, corresponding to position 278 of SEQ ID NO: 9, and is either A or G. This application is for identifying or assisting in the identification of cadmium content in wheat and / or for wheat breeding.

[0006] In the above applications, the substance may be B1), B2), or B3). B1) The substance described is a primer composition for amplifying wheat genomic DNA fragments including the SNP described above. B2) The substance is a PCR reagent containing the primer composition described in B1). B3) The substance is a kit containing the primer composition described in B1) or the PCR reagent described in B2).

[0007] The present invention also provides a method for identifying or assisting in the identification of wheat cadmium content traits. The method may include detecting the genotype of SNPs in the genome of the wheat to be tested, and identifying or assisting in the identification of wheat cadmium content traits based on the genotypes. The SNPs may be TaCd2-1, TaCd2-2, and TaCd3 as described above.

[0008] In some embodiments of the present invention, the cadmium content of wheat grains with the GGCCAA genotype is higher than that of wheat grains with the non-GGCCAA genotype. The non-GGCCAA genotype wheat includes wheat with the CCGGAA genotype, wheat grains with the GGCCGG genotype, and wheat with the CCGGGG genotype. The GGCCAA genotype wheat is homozygous for TaCd2-1 (G), TaCd2-2 (C), and TaCd3 (A). Wheat with the CCGG genotype is homozygous for TaCd2-1 (G), TaCd2-2 (C), and TaCd3 (G); wheat with the CCGGAA genotype is homozygous for TaCd2-1 (C), TaCd2-2 (G), and TaCd3 (A); and wheat with the CCGGGG genotype is homozygous for TaCd2-1 (C), TaCd2-2 (G), and TaCd3 (G).

[0009] In some embodiments of the present invention, there is no statistically significant difference in cadmium content in wheat grains of the non-GGCCAA genotype, the CCGGAA genotype, the GGCCGG genotype, and the CCGGGG genotype.

[0010] The “cadmium content in wheat grains” is a comparison conducted under comparable conditions. “Comparable conditions” refer to the same or similar environmental conditions and agronomic practices used to make meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices significantly contribute to or explain any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water, humidity, soil, and nutrients (e.g., nitrogen and phosphorus).

[0011] This invention also provides the application of the methods described above in wheat breeding.

[0012] This invention also provides a method for wheat breeding, the method comprising detecting TaCd2-1, TaCd2-2 and TaCd3 as described above in the wheat genome, and selecting any one of the following wheat varieties as a parent for breeding: 1) Wheat in which TaCd2-1 is homozygous for G, TaCd2-2 is homozygous for C, and TaCd3 is homozygous for G; 2) Wheat in which TaCd2-1 is homozygous for C, TaCd2-2 is homozygous for G, and TaCd3 is homozygous for A; 3) Wheat in which TaCd2-1 is homozygous for C, TaCd2-2 is homozygous for G, and TaCd3 is homozygous for G.

[0013] In the above applications and methods, the substance used to detect SNP polymorphisms, genotypes, or haplotypes can be a nucleotide type at the SNP site in the wheat genome determined by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP microarrays. The SNP microarrays include microarrays based on nucleic acid hybridization reactions, microarrays based on single-base extension reactions, microarrays based on allele-specific primer extension reactions, microarrays based on one-step reactions, microarrays based on primer ligation reactions, microarrays based on restriction endonuclease reactions, microarrays based on protein-DNA binding reactions, and microarrays based on fluorescent molecule-DNA binding reactions.

[0014] This invention also provides a product for detecting polymorphisms, genotypes, or haplotypes of SNPs in the wheat genome, wherein the SNP may be the SNP described above, and the product contains the substance described above, wherein the product is as follows: D1), D2), or D3). D1) The product is a primer composition for amplifying wheat genomic DNA fragments including the SNP sites. D2) The product is a PCR reagent containing the primer composition described in D1). D3) The product is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0015] In the above applications and products, the primer composition used for detection may consist of TaCd2-1 primer pairs, TaCd2-2 primer pairs, and / or TaCd3 primer pairs; the TaCd2-1 primer pairs consist of single-stranded DNA from SEQ ID NO: 1 and SEQ ID NO: 2, the TaCd2-2 primer pairs consist of single-stranded DNA from SEQ ID NO: 3 and SEQ ID NO: 4, and the TaCd3 primer pairs consist of single-stranded DNA from SEQ ID NO: 5 and SEQ ID NO: 6.

[0016] In the above applications, the primer composition may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Markers include, but are not limited to, dyes; radioactive markers, such as 32P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The marker can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative) or, optionally, charge-neutral. The marker can include a nucleic acid sequence or a protein sequence or a combination thereof, provided that the sequence containing the marker is detectable. In some embodiments, nucleic acids are detected directly without a marker (e.g., direct sequence reading).

[0017] The present invention also provides a PCR reagent containing the primer composition described above for detecting polymorphisms or genotypes of SNP sites in the wheat genome.

[0018] The present invention also provides a kit containing the PCR reagents described above for detecting polymorphisms or genotypes of SNP sites in the wheat genome.

[0019] The present invention also provides a nucleic acid molecule, wherein the nucleotide sequence of the nucleic acid molecule may be SEQ ID NO: 7, SEQ ID NO: 8 and / or SEQ ID NO: 9.

[0020] The indicators for wheat breeding mentioned above include wheat cadmium content.

[0021] The cadmium content in wheat mentioned above specifically refers to the cadmium content at the wheat's maturity stage.

[0022] Furthermore, the purpose of the wheat breeding includes developing wheat with low cadmium content (wheat with cadmium content lower than that of the parents).

[0023] In some embodiments, the cadmium content of wheat refers to the cadmium content of wheat grains.

[0024] In the above applications and methods, the wheat can be a homozygous wheat inbred line.

[0025] In the applications and methods described above, homozygous wheat inbred lines can be selected as parents for breeding.

[0026] This invention, through the study of the single-gene haplotype and combined haplotype effects of two wheat cadmium transporter genes, TaCd2 and TaCd3, has discovered a combined haplotype that can be used to screen for high-cadmium varieties. This provides a genetic basis for the rapid screening of wheat germplasm at high cadmium risk and for early warning of high cadmium risk in wheat cultivation on cadmium-contaminated land, and has important theoretical value and application prospects for ensuring my country's food security. Attached Figure Description

[0027] Figure 1 This is a frequency distribution map of cadmium content in resource germplasm.

[0028] Figure 2 It refers to the cadmium content of haplotypes TaCd2 and TaCd3 in the grains.

[0029] Figure 3 It refers to the cadmium content of the four combined haplotypes in the grain. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0032] The 145 wheat samples in the following examples and the 107 wheat samples in Table 2 are disclosed in Supplemental Table 1 of the following literature: Chenyang Hao#, Chengzhi Jiao#, Jian Hou, Tian Li, Hongxia Liu, Yuquan Wang, Jun Zheng, Hong Liu, Zhihong Bi, Fengfeng Xu, Jing Zhao, Lin Ma, Yamei Wang, Uzma Majeed, Xu Liu, Rudi Appels, Marco Maccaferri, Roberto Tuberosa, Hongfeng Lu*, Xueyong Zhang*. Resequencing of 145 landmark cultivars reveals asymmetric sub-genome selection and strong founder genotype effects on wheat breeding in China. Molecular Plant, 2020, 13: 1733-1751. These materials are publicly available from the Institute of Botany, Chinese Academy of Sciences. This biological material is intended solely for the replication of experiments of this invention and should not be used for any other purpose.

[0033] Example 1: Using three SNPs, TaCd2-1, TaCd2-2, and TaCd3, to identify the cadmium content in wheat grains. 1. Cultivation and content determination of wheat germplasm resources 1.1 Determination of soil cadmium content Weigh 0.1000 g of sieved soil into a polytetrafluoroethylene digestion tube, and add 1 mL of hydrofluoric acid, 1 mL of aqua regia (concentrated hydrochloric acid: concentrated nitric acid = 3:1), and 1 mL of perchloric acid in sequence. Cap and digest at 150℃ for 5 h. Open the cap halfway through digestion to allow the acidic fumes to completely evaporate. After cooling to room temperature, add 2 mL of nitric acid and continue heating for 3 h until the soil sample is completely clear. Add 2 mL of nitric acid and 10 mL of distilled water, cap, and reconstitute for 4 h to dissolve the residue. Filter through a 0.22 μm filter membrane into a 10 mL plastic centrifuge tube. The soil control is the Tibetan Soil Composition Analysis Standard Material (Institute of Geophysical and Geochemical Exploration, GBW08302).

[0034] 1.2 Cultivation of Wheat Germplasm Resources 145 wheat microcore germplasm resources were planted in Weihui City, Xinxiang City, Henan Province in the winter of 2023, with soil cadmium content of 1.755±0.323 mg / kg.

[0035] 1.3 Determination of Cadmium Content in Wheat Grains (1) Select 30 wheat grains harvested in the spring of 2024 after they have matured. After dehulling by hand, put them into a 5 mL centrifuge tube and add 1 steel ball for grinding. Program: 1800 rpm, 3 cycles, 55 s each time, 35 s rest.

[0036] (2) Accurately weigh 0.2000 g of wheat seeds and put them into a digestion tube.

[0037] (3) Add 1 mL of high-purity nitric acid to the digestion tube (operate in a fume hood), place a bent funnel inside, cover it with a plastic bag, and let it digest overnight in a cold environment.

[0038] (4) On the second day, observe the cold digestion to ensure that digestion is complete. You can add some nitric acid as appropriate. Set the digestion oven to 200°C and digest for 9 hours until the liquid is clear.

[0039] (5) After removing it and letting it cool, add distilled water to the clear liquid after digestion and make up to 15 mL using a volumetric immersion tube.

[0040] (6) Shake well, filter through a 0.22 μm filter membrane into a 10 mL plastic centrifuge tube, and determine the cadmium content using an inductively coupled plasma mass spectrometer (ICP-MS).

[0041] For each batch, three positive controls were set up using wheat flour composition analysis standard material (Institute of Geophysical and Geochemical Exploration, GBW(E)100493), and two blank controls were set up to remove acid interference.

[0042] 2. Identification of non-synonymous mutation sites in wheat cadmium transporter genes: Primers were designed upstream and downstream of the nonsynonymous mutation and PCR amplification was performed using KOD FX enzyme. The primer sequences are shown in Table 1, and the PCR procedure is as follows: (1) Preparation of reaction solution: 2×PCR Buffer for KOD FX25 μL dNTPs 10 μL Forward Primer 1.5 μL Reverse Primer 1.5 μL Template DNA≤50ng (plasmid) / ≤200 ng (DNA / cDNA) KOD FX Enzyme 1 μL Add ddH2O to bring the volume to 50 μL. (2) PCR reaction procedure 94℃ for 2 min Perform the following three steps 40 times. 98℃ for 10 seconds 57℃ for 30 seconds 68℃ 30 s After the loop ends, extend for 7 minutes.

[0043] 68℃ for 7 min 4℃∞ The results of PCR amplification were subjected to Sanger sequencing, which was performed by BGI Genomics.

[0044] Table 1 Primer Sequences

[0045] The PCR product of the TaCd2-1 primer set is SEQ ID NO: 7 (526bp). The 518690092 (TaCd2-1) site corresponds to position 320 of SEQ ID NO: 7, which is either C or G (represented by "S"). The polymorphism is CC or GG, as detailed below: 5'--3'.

[0046] The PCR product of the TaCd2-2 primer set is SEQ ID NO: 8 (522bp). The 518690690 (TaCd2-2) site corresponds to position 233 of SEQ ID NO: 8, which is either C or G (represented by "S"). The polymorphism is CC or GG, as detailed below: 5'--3'.

[0047] The PCR product of the TaCd3 primer set is SEQ ID NO: 9 (539bp). The 442075003 position (TaCd3) corresponds to position 278 of SEQ ID NO: 9, which is either A or G (indicated by "R"). The polymorphism is AA or GG, as detailed below: 5'--3'.

[0048] 3. Genotypic analysis of wheat cadmium transporter genes The TaCd2 and TaCd3 genes were analyzed to investigate the relationship between natural variation and phenotype. One-way ANOVA was used to compare the statistical differences in grain cadmium content among different combined haplotypes using data from co-genotypes. P-values ​​were calculated using Tukey's test. A p-value less than 0.05 was defined as statistically significant; a p-value less than 0.01 was defined as highly statistically significant.

[0049] III. Research Results 1. Determination of cadmium content in resource germplasm The cadmium content of the seeds of resource germplasm planted in cadmium-contaminated soil was determined, and the results are as follows: Figure 1 As shown, cadmium content was successfully detected in the grains of 107 out of 145 wheat samples, with cadmium concentrations ranging from 0.09 to 0.72 mg / kg. Figure 1The horizontal axis represents the interval mean. 0.1 represents the cadmium concentration in the grain of the germplasm that is ≥0.075 and <0.125 mg / kg, 0.15 represents the zinc concentration in the grain of the germplasm that is ≥0.125 and <0.175 mg / kg, and so on.

[0050] 2. Identification of non-synonymous mutation sites in wheat TaCd2 and TaCd3 Among the 107 wheat germplasm populations where cadmium content was detected, the wheat cadmium transporter TaCd2 (TraesCS4B03G0683300, Chr4B) had two non-synonymous mutation sites (Chr4B:518690092; Chr4B:518690690), named TaCd2-1 and TaCd2-2, respectively, and TaCd3 (TraesCS4D03G0643500, Chr4D) had one non-synonymous mutation site (Chr4D:442075003) (Table 2), named TaCd3. The non-synonymous mutation sites were identified by Sanger sequencing using primers, and the sequencing results are shown in Table 2. The reference genome version for labeling nonsynonymous mutation sites in wheat is Chinese Spring v2.1 (https: / / phytozome-next.jgi.doe.gov / report / gene / Taestivumcv_ChineseSpring_v2_1 / TraesCS4B03G0683300).

[0051] Table 2. Nonsynonymous loci, haplotypes, genotypes, and cadmium content of 107 wheat resource population varieties.

[0052] 3. Single-gene haplotype analysis of wheat TaCd2 and TaCd3 The two genes were genotyped, and the results are shown in Tables 3 and 4. TaCd2 has two haplotypes: TaCd2Hap1 (GC) and TaCd2Hap2 (CG). The genotype corresponding to TaCd2Hap1 is GGCC (TaCd2-1 is homozygous for G and TaCd2-2 is homozygous for C), representing two SNP combinations: TaCd2-1 genotype GG and TaCd2-2 genotype CC. The genotype of TaCd2Hap2 is also a combination of two SNPs: TaCd2-1 genotype CC and TaCd2-2 genotype GG. TaCd3 has two genotypes: TaCd3Hap1 (A) and TaCd3Hap2 (G). TaCd3Hap1 (A) is the genotype of TaCd3 with the genotype AA, and TaCd3Hap2 (G) is the genotype of TaCd3 with the genotype GG.

[0053] Based on haplotypes, the relationship between different haplotypes of the two genes and cadmium content was analyzed. The results showed that: TaCd2Hap1 contained 24 germplasms with a grain cadmium content of 0.303±0.129 mg / kg; TaCd2Hap2 contained 83 germplasms with a grain cadmium content of 0.267±0.088 mg / kg; TaCd3Hap1 contained 36 germplasms with a grain cadmium content of 0.281±0.115 mg / kg; and TaCd3Hap2 contained 71 germplasms with a grain cadmium content of 0.272±0.080 mg / kg. The results indicate that the haplotypes of both single genes had no significant effect on grain cadmium accumulation. Figure 2 ).

[0054] Table 3. TaCd2 haplotypes and cadmium content in grains

[0055] Table 4. TaCd3 haplotypes and cadmium content in seeds

[0056] 4. Analysis of haplotypes and genotypes of wheat TaCd2 and TaCd3. Combining TaCd2 and TaCd3, we can classify them into four haplotypes: MHap1, MHap2, MHap3, and MHap4 (Table 5), which correspond to the following four genotypes: GGCCAA (homozygous for G in TaCd2-1, C in TaCd2-2, and A in TaCd3), GGCCGG (homozygous for G in TaCd2-1, C in TaCd2-2, and G in TaCd3), CCGGAA (homozygous for C in TaCd2-1, G in TaCd2-2, and A in TaCd3), and CCGGGG (homozygous for C in TaCd2-1, G in TaCd2-2, and G in TaCd3).

[0057] Cadmium content analysis of the combined haplotypes TaCd2 and TaCd3 revealed the following values: MHap1 had a cadmium content of 0.399 ± 0.202 mg / kg; MHap2 had a cadmium content of 0.271 ± 0.079 mg / kg; MHap3 had a cadmium content of 0.258 ± 0.074 mg / kg; and MHap4 had a cadmium content of 0.273 ± 0.095 mg / kg. The cadmium content of MHap1 was significantly higher than that of the other three combined haplotypes. Figure 3 (This can be used to identify wheat germplasm at high cadmium risk).

[0058] Table 5. Four combined haplotypes and genotypes and cadmium content in grains.

[0059] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. An application for detecting polymorphisms, genotypes, or haplotypes of SNPs in the wheat genome, characterized in that, The SNPs are TaCd2-1, TaCd2-2, and TaCd3; TaCd2-1 is an SNP in the wheat genome, corresponding to position 320 of SEQ ID NO: 7, and is either C or G. TaCd2-2 is a SNP in the wheat genome, corresponding to position 233 of SEQ ID NO: 8, and it is either C or G; TaCd3 is a SNP in the wheat genome, corresponding to position 278 of SEQ ID NO: 9, and is either A or G; the application is for the identification or auxiliary identification of wheat cadmium content and / or wheat breeding applications.

2. The application of the substance for detecting SNPs in the wheat genome according to claim 1, characterized in that, The substance is B1), B2), or B3): B1) The substance described is a primer composition for amplifying wheat genomic DNA fragments including the SNP described above. B2) The substance is a PCR reagent containing the primer composition described in B1). B3) The substance is a kit containing the primer composition described in B1) or the PCR reagent described in B2).

3. A method for identifying or assisting in the identification of cadmium content in wheat, characterized in that, The method includes detecting the genotype of the SNP described in claim 1 in the genome of the wheat to be tested, and identifying or assisting in the identification of the cadmium content of the wheat based on the genotype, wherein the SNP is TaCd2-1, TaCd2-2 and TaCd3 as described in claim 1.

4. The application of the method of claim 3 in wheat breeding.

5. A method for wheat breeding, characterized in that, The method includes detecting TaCd2-1, TaCd2-2, and TaCd3 as described in claim 1 in the wheat genome, and selecting any one of the following wheat varieties as a parent for breeding: 1) Wheat in which TaCd2-1 is homozygous for G, TaCd2-2 is homozygous for C, and TaCd3 is homozygous for G; 2) Wheat in which TaCd2-1 is homozygous for C, TaCd2-2 is homozygous for G, and TaCd3 is homozygous for A; 3) Wheat in which TaCd2-1 is homozygous for C, TaCd2-2 is homozygous for G, and TaCd3 is homozygous for G.

6. A product for detecting SNP polymorphisms, genotypes, or haplotypes in the wheat genome, characterized in that, The SNP is the SNP of claim 1, the product contains the substance of claim 1, and the product is as follows: D1), D2), or D3). D1) The product is a primer composition for amplifying wheat genomic DNA fragments including the SNP sites. D2) The product is a PCR reagent containing the primer composition described in D1). D3) The product is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

7. The application according to claim 2 and the product according to claim 6, characterized in that, The primer composition used for detection consists of TaCd2-1 primer pair, TaCd2-2 primer pair and / or TaCd3 primer pair; the TaCd2-1 primer pair consists of single-stranded DNA of SEQ ID NO: 1 and SEQ ID NO: 2, the TaCd2-2 primer pair consists of single-stranded DNA of SEQ ID NO: 3 and SEQ ID NO: 4, and the TaCd3 primer pair consists of single-stranded DNA of SEQ ID NO: 5 and SEQ ID NO:

6.

8. A PCR reagent containing the primer composition of claim 7 for detecting polymorphisms or genotypes of SNP sites in the wheat genome.

9. A kit containing the PCR reagent of claim 8 for detecting polymorphisms or genotypes of SNP sites in the wheat genome.

10. A nucleic acid molecule, characterized in that, The nucleotide sequences of the nucleic acid molecule are SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

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